1. Field of the Invention
This invention relates to an autoradiographic process. Particularly, the invention relates to an autoradiographic process for obtaining each locational information on at least two kinds of radioactively labeled substances which are contained in a sample selected from the group consisting of tissue of an organism and a medium containing tissue of an organism and/or substances originating from an organism.
2. Description of Prior Art
There has been heretofore known an autoradiographic process termed "autoradiography" or "radioautography" comprising the steps of: introducing a radioactively labeled substance into an organism; placing the organism or a part of tissue of the organism (i.e., a sample or specimen of animals, plants, fishes, etc.) on a radiographic film such as a high sensitivity type X-ray film for a given period of time to expose the film thereto; and obtaining the locational information on the radioactively labeled substance in the specimen from the resolved pattern of the film. The autoradiography has been utilized, for example, to investigate the pathway and state of metabolism, absorption, and excretion of the substance introduced into animals, plants, etc., in detail.
Recently, the autoradiography has been utilized for obtaining locational information on the radioactively labeled substances present on a medium containing radioactively labeled tissue of an organism or the radioactively labeled substances originating from an organism. For instance, there is known an autoradiography comprising the steps of: labeling organism-originating biopolymers such as proteins or nucleic acids with a radioactive element; resolving the mixture of the radioactively labeled biopolymers, derivatives thereof, or cleavage products thereof on a gel support (medium) through a resolving process such as gel electrophoresis: placing the gel support on a high sensitivity X-ray film for a given period of time to expose the film to the gel support, developing the film, obtaining the locational information of the radioactively labeled substances from the developed film, and then performing the identification of the polymeric substances, determination of molecular weight of the polymeric substances and isolation of the polymeric substances based on the obtained locational information.
Further, in order to anlalyze the pathway of an organism, an autoradiography utilizing the double-labeling method (i.e., double-tracer method) in which two kinds of radioactive isotopes are used has been proposed. The autoradiography comprises the steps of introducing each of radioactively labeled substances labeled with at least two kinds of radioactive isotopes different from each other into an organism and obtaining autoradiogram corresponding to each of the radioactively labeled substances (i.e., each isotope). Thus, this process is utilized to anlyze metabolism and chemical reaction in the organism.
As radiographic films employed for the autoradiography utilizing the double-labeling method, Japanese Patent Publication No. 47(1972)-45540 discloses a silver halide photographic photosensitive material for color autoradiography employable for distinguishably recording the distribution images of tritium (.sup.3 H) and other radioactive isotopes. The material comprises two silver halide emulsion layers each of which contains a coupler having hue different from each other and thicknesses of which are adjusted to enable the distinguishable recording.
In the autoradiography according to the conventional radiographic method, however, it is extremely difficult to obtain the autoradiogram corresponding to each isotope of a sample which is multiply labeled with different radioactive isotopes. Even if the radiographic film as mentioned above is employed for the multiply labeled sample, the resultant autoradiograms are visibly recorded on one radiographic film as color images having hues different from each other so that investigators are required to examine and analyze the each autoradiogram only by difference of the hues. Further, different autoradiograms are recorded on one radiographic film so that locational information on radioactively labeled substances can not accurately be obtained.
For this reason, a double-autoradiography (i.e., two nuclides labeled autoradiography) utilizing difference of half-lives of radioactive isotopes has been recently employed. This process comprises the steps of: introducing two kinds of radioactively labeled substances which are labeled with two nuclides having half-lives greatly different from each other (e.g., .sup.123 I: half-life of about 13 hours, .sup.14 C: half-life of about 5,730 years) into an organism; taking a sample from the organism after a certain period of time in order to examine distribution of each of the radioactively labeled substances in the organism; placing the sample on a radiographic film; immediately subjecting the film to autoradiography to obtain an image of the two kinds of radioactively labeled substances which are labeled with the two nuclides on the radiographic film; allowing the sample to stand for a long time (i.e., until the nuclide having a short half-life almost disappears); subjecting the sample to autoradiography similar to the above autoradiography to obtain an image of the radioactively labeled nuclide of a long half-life on the radiographic film; and performing a subtraction processing which comprises subtracting the latter image of the radioactively labeled substance obtained by autoradiography (i.e., an image showing distribution of the radioactively labeled nuclide of a long half-life in the sample) from the former image of the radioactively labeled substances obtained by the autoradiography to obtain an image showing distribution of the radioactively labeled nuclide of a short half-life in the sample.
In this process, although each of the images obtained above has high accuracy, a standing period required for allowing the nuclide having a short half-life to almost disappear is very long (e.g., in the combination of .sup.123 I and .sup.14 C, about two months are required), which retards progress on the study. Further, since the relationship between the density of the image formed on the silver halide film (i.e., blackening image) and the radiation amount does not show simple linearity, the above subtraction processing is required to perform using a complicated formula of relation.
Further, such autoradiography using a radiographic film (i.e., silver halide photosensitive photographic film) is not free from several drawback in the practical use.
In order to obtain the autoradiogram, a procedure of placing a sample containing a radioactively labeled substances (which are labeled with radioactive isotopes) on a radiographic film such as a high-sensitivity X-ray film for a given period of time and exposing the film to the substances is performed. In this procesure, the exposing procedure requires a long period of time (ten hours to several days). This is because samples for the autoradiography generally do not have high radioactivity. Further, the exposing procedure must be performed at a low temperature (e.g., 0 to -80.degree. C.). The reason is that a latent image of the photosensitive silver salt formed on the film is some-times faded to become an image which is not developable or that the latent image is chemically fogged by various substances harmful to silver salt which migrate from the sample into the film at relatively high temperatures. Such chemical fog results in difficulty of obtaining locational information on the radioactively labeled substances with high accuracy. Thus, the exposure ought to be carried out at a low temperature to reduce the chemical fog. Further, in order to prevent lowering of the image quality originating from the chemical fog, the example containing the radioactively labeled substances should be superposed on the radioactive film in the drying condition during the exposure, and therefore the example is generally required to be dried or to be packaged within a polymer film.
The photosensitive silver salt of a radiographic film has a drawback that it is further sensitive not only to the chemical irritation but also to physical impetus caused in such operation as transfer or setting of the film, this drawback brings about difficulty in the autoradiographic procedure and decreases accuracy thereof. In more detail, the radiographic film is likely brought into contact with samples, hands of the operator and tools in the handling, and such physical pressure arising from these contacts causes production of the physical fog on the radiographic film. The physical fog is also a cause of the decrease of accuracy in the autoradiography. For this reason, the handling of a radiographic film requires well-trained skill and great caution to avoid the production of the physical fog on the radiographic film, and such requirement of careful handling increases complexity in the autoradiographic procedure.
Further, certain natural radioactive substances contained in the sample in addition to the radioactively labeled substance take part in the exposure of the radiographic film because the exposure is carried out, as described above, for a long period of time in the conventional autoradiography. Thus, the influence of the natural radioactive substance further reduces the accuracy of locational information of the radioactively labeled substances, and this is an additional drawback. In order to remove the troublesome noise brought about by the natural radioactive substances, parallel experiments using control samples and a method for optimization of the exposure time have been employed, but these procedures include increased experimental runs for the parallel experiments and requires preliminary experiments to determine the preferable exposure time, and thus the drawback arising from the complicated procedures is not avoidable as whole.
In order to remove the drawbacks of the conventional autoradiography using the above photosensitive silver halide film, use of a stimulable phosphor sheet (i.e., radiation image storage panel) having a phosphor layer containing a stimulable phosphor as the photosensitive material has been already proposed in EP-A-0,111,154.
Further, to solve the problems observed in the autoradiography utilizing the double-labeling method, use of a stimulable phosphor sheet or a composite thereof having plural phosphor layers containing a stimulable phosphor as the photosensitive material has been already proposed in Japanese Patent Provisional Publication No. 62(1987)-93679.
Although the latter invention can effectively solve the above problems of the conventional autoradiography utilizing the double-labeling method, it is necessary to prepare plural stimulable phosphor layers (composite or plural stimulable phosphor sheets) having thicknesses different from each other, the number of the phosphor layers corresponding to the number of nuclides used. Further, the invention enables to obtain radiation images corresponding to plural nuclides by one read-out operation, while it is not easy to determine the conditions for separating these radiation images.